1. Field of the Invention
The present invention relates to a labeled PCR product used for a DNA chip, and also to a method for labeling sample nucleic acid and a liquid composition used for nucleic acid labeling. The present invention further relates to a highly sensitive detection method for specimen.
2. Related Background Art
As typified by the human genome project, genes of various types of organisms have been clarified. Association of genes with mechanisms of vital activities, diseases, constitutions, and other factors have successively been examined. As a result, it has been found out that by determining the presence or absence of a specific gene or its expression level (abundance), diseases can be characterized or classified in more detail and effective therapeutic methods therefor can be chosen.
Many methods for determining the presence or absence of a specific gene or its abundance contained in a sample have been proposed over a long period of time. Among them, a method that chooses a specific partial sequence of a target gene or nucleic acid and examines the presence or absence of the partial sequence or the amount in a sample is widely used to know the presence or abundance of such a gene because of its wide applicability. More specifically, this method comprises preparing a nucleic acid (probe) corresponding to a complementary strand of the chosen partial sequence and detecting hybridization between the sample and the probe by any means, so as to determine the presence or absence of the nucleic acid sequence in the sample.
Detection of a specific nucleic acid using hybridization can be carried out either in a solid phase or in a liquid phase. When hybridization is carried out on a solid substrate, a typical method is to immobilize or adsorb a probe on the solid substrate, and then add thereto a sample labeled with a certain labeling substance enabling detection, so that detection is carried out by measuring the signal of the labeling substance on the solid substrate. In particular, a representative form used in solid-phase hybridization is a chip in which one or more probes are immobilized or adsorbed on a planar substrate such as a glass or metal, or a bead carrying a probe immobilized on the surface of a fine particle. The reason why solid-phase hybridization is preferable is that B/F separation is easy, the detection region can physically be made very small whereby high sensitivity is expected, plural types of probes can be separated physically thereby enabling simultaneous detection of multi-items, and handling and application area easy because of the solid phase.
As disclosed in U.S. Pat. No. 6,410,229, for example, a labeled sample nucleic acid is reacted with oligo DNA synthesized on a planar substrate, and the hybridization is measured by fluorescence detection, so as to detect the presence or absence of a specific nucleic acid in a sample or the amount thereof. Japanese Patent Application Laid-Open No. 2001-128683 discloses preparation of a DNA array using a substrate provided with amino groups to detect a 22-mer single-stranded labeled DNA.
In the aforementioned methods for a detecting specific nucleic acid using hybridization, the sample nucleic acid should be labeled. One method for incorporating a labeling substance into a sample is to add a labeled deoxynucleotide (e.g. Cy3-dUTP) in polymerase chain reaction (PCR).
In that case, four types of deoxynucleotides (dATP, dCTP, dGTP, and dTTP; generically called dNTP) and a labeled deoxynucleotide (e.g. Cy3-dUTP) are prepared for the substrates in PCR where the final concentrations of all the dNTPs may be made the same. In Japanese Patent No. 3,001,919, for example, one of the four types of substrate nucleotides is partly replaced by a fluorescence-labeled nucleotide to label the product.
As described above, a detection method utilizing solid-phase hybridization is advantageous in that it is more sensitive than other detection methods. However, the need for detection of a trace amount of nucleic acid is exceeding it, and thus, further improvements of such detection method are required for realizing much higher specific detection with higher sensitivity. Various attempts have been made to improve detection performance. One method is to design a probe to increase the Tm value of the probe-target hybrid, so as to enhance the ability of the probe to bind the target. Another method is labeling of the target with a substance emitting a strong signal, or intensify the labeling substance itself using several labeling substances (sensitizers).
However, the method of enhancing the binding ability of a probe to the target may cause non-specific adsorption or binding, and may significantly decrease specificity in some cases. Thus, the advantage of such a method is limited. On the other hand, the method of amplifying the signal of a labeling substance may significantly decrease the S/N ratio or quantitativeness in some cases. Thus, the advantage of such a method is also limited.
Accordingly, it has been strongly desired to develop a method of efficient incorporation of a labeling substance into a sample which enables more efficient nucleic acid detection. Conventional methods of incorporating a labeling substance into a sample described in the above, in which a labeled deoxynucleotide is added during PCR, has a problem that not so much labeling substance is incorporated into the amplified product since a large amount of non-labeled deoxynucleotide is present as a substrate in PCR, and the detection amount by hybridization may not correspond to the amount of the synthesized amplified product.
In the case of a labeling method using dNTPs in the same final concentrations, if the proportion of the labeled deoxynucleotide is increased to a certain extent, the labeling substance is efficiently incorporated. However, labeled deoxynucleotides are extremely expensive, and thus, the application range thereof may be limited.